PSEUDO- AND NON-PEPTIDE BRADYKININ RECEPTOR ANTAGONISTS

Abstract: The invention provides bradykinin antagonist compounds wherein many (or all) of the peptide bonds of bradykinin are eliminated to yield compounds which specifically compete with bradykinin for binding to the bradykinin receptor. More particularly, the invention relates to compounds having, in appropriate spatial arrangement, two positively charged moieties flanking a hydrophobic organic moiety and a moiety which mimics a beta turn conformation.

Description: PSRTΪDO- AND N N-PEPTTDE BR A nYKTNTN RECEPTOR ANTAGONISTS TECHNICAL FTEIT) This invention relates to compounds which specifically compete with native bradykinin for binding to the bradykinin B2 receptor and to compounds which specifically compete with des Arg^-bradykinin for the bradykinin Bi receptor. The invention also relates to pharmaceutical compositions and methods for using the compounds to antagonize the effect of bradykinin in mammals, including humans. More particularly, the invention relates to compounds having, in appropriate spatial arrangement, two positively charged moieties flanking a hydrophobic organic moiety and a moiety which mimics a beta turn conformation. These compounds interact with the bradykinin B2 receptor in such a way to specifically compete with the binding of native bradykinin. Corresponding compounds which lack the positively charged moiety at one end of the molecule interact with the bradykinin Bi receptor in such a way to specifically compete with the binding of des Arg^-bradykinin. BACKGROUND ART Bradykinin (BK) is a linear nonapeptide produced endogenously in humans and other mammals as a result of the activity of kallikreins, a group of proteolytic enzymes present in most tissues and body fluids, on kininogens. Once released, kinins produce many physiological responses, including pain and hyperalgesia by stimulating C- and A-fibers in the periphery. There is also considerable evidence that kinins contribute to the inflammatory response. Native bradykinin has the amino acid structure shown below: Arg - Pro - Pro - Gly - Phe - Ser - Pro - Phe - Arg 1 2 3 4 5 6 7 8 9 Bradykinin, and its physiologically important related peptides kalladin (Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiological actions which qualify them as mediators of inflammatory reactions, hypotensive states, and pain. Bradykinin is overproduced in pathological conditions such as septic shock, anaphylaxis, rhinitis, asthma, inflammatory bowel disease, and certain other conditions including acute pancreatitis, post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, and angioneurotic edema. The production of bradykinin from the plasma results in pain at the site of the pathological condition, and the overproduction intensifies the pain directly or via bradykinin- induced activation of the arachidonic acid pathway which produces prostaglandins and leukotrienes, the more distal and actual mediators of inflammation. In addition to its proinflammatory effects, bradykinin is a vasodilator. Because of its concomitant ability to lower blood pressure, bradykinin has been implicated in the pathogenesis of several shock syndromes, particularly septic or endotoxic shock. Bradykinin is also a potent bronchoconstrictor in animals and asthmatic subjects and it has been implicated as a contributor to the pathogenesis of airway inflammatory conditions such as allergic asthma and rhinitis. As a result of the implication that increased levels of bradykinin may play a part in a number of pathological conditions, considerable research has been aimed toward the derivation of bradykinin receptor antagonists as potential therapeutic agents. A bradykinin receptor antagonist is expected to possess a number of desirable biological effects in the treatment, for example, of pain and inflammation, septic shock, airway disorders such as asthma, burn pain, pancreatitis, angioedema, certain nervous system disorders, chronic inflammation such as rheumatoid arthritis and inflammatory bowel disease, rhinitis, and allergy. Several non-peptide, non-specific and non-selective antagonists of one or more of the biological activities of bradykinin have been described among compounds as diverse as analgesics and anti-inflammatory substances, which act via the prostaglandin system and not directly on bradykinin receptors. These are antihistamines, bradykinin-antibodies, benzodiazepine derivatives, high molecular weight ethylene oxide polymers, gallic acid esters, and serotonin inhibitors. None of these compounds or classes of compounds specifically inhibit the effects of bradykinin. Heptyl esters of various amino acid-containing substances, such as single basic amino acids the dipeptide Phe-Gly, and analogs of C-terminal peptide fragments of bradykinin (i.e., Pro-Phe-Arg) have been reported as anti-bradykinin substances. When tested in bradykinin assay systems, they prove to be weak partial agonists/antagonists, depending on the dose, with little specificity for inhibiting bradykinin action. Several research groups have prepared bradykinin receptor antagonists. The first antagonists of bradykinin were discovered by Stewart and Vavrek. U.S. Patent No. 4,801,613 and 4,693,993 (which references are incorporated in their entirety herein) disclose a series of bradykinin antagonists wherein the L-Pro at the 7-position of the peptide hormone bradykinin or other substituted analogs of bradykinin is substituted with an aromatic amino acid of the D-configuration which converts bradykinin agonists into bradykinin antagonists. The specific L-Pro substitutions are selected from the group consisting of D-Nal, D-PNF, D-Phe, D-Tyr, D-Pal, D-OMT, D-Thi, D-Ala, D-Trp, D-His, D-Homo-Phe, D-Phe, pCl-D-Phe (CDF), D-Phg, D-Val, D-Ile, D-Leu, and MDY. Typically, these bradykinin antagonist peptides had Kj values in the range of 20-80 nM in guinea pig ileum (Stewart, et al., In Bradykinin Antagonists (1991) Burch, R.M., Marcel Dekker, New York). Subsequently, several classes of bradykinin antagonist peptides with 600- 1000-fold greater potency in the guinea pig ileum preparation have been disclosed. Published European Patent Application No. 0413 277 Al to Hoechst A.G. discloses bradykinin antagonists containing the aromatic amino acid D-Phe at position 7 but containing unnatural amino acids at position 8 which impart increased potency. Published European Patent Application No. 0 370453 A2 to Hoechst A.G. discloses bradykinin antagonists containing a D-amino acid (D-Tic) at position 7. A more recent series of bradykinin receptor antagonist peptides lacks the D-aromatic amino acid at position 7 which was believed to be critical to the activity of the earlier described antagonists of the endogenous neuropeptide. As described in published PCT application WO 92/18156 and WO 92/18155 (which references are incorporated in their entirety herein) this group of compounds have a general bradykinin antagonist structure wherein the L-Pro at position 7 is substituted with hydroxyproline ether and thioether derivatives (termed D-Hype) and the L-Phe at position 8 can additionally be substituted with hydroxyproline ethers and thioethers derivatives (Hype), Tic or Oic. The bradykinin antagonist peptides referred to above exert their activity by blocking the bradykinin B2 receptor. A second bradykinin receptor, the B i receptor, is not expressed to any significant degree in healthy tissue, but its expression is upregulated during persistent inflammatory hyperalgesia. This receptor is activated by des Arg^-kalladin and des Arg 9 "bradykinin, a proteolytic degradation product of bradykinin. It is believed to play an important role in the maintenance of hyperalgesia in chronic inflammatory conditions (Dray, A. and Perkins, M., TINS (1993) i£(3):99-103). [Des-Arg9] analogs of bradykinin B2 receptor antagonist peptides of the type described above, bind to the Bi receptor and have been shown to reverse or prevent hyperalgesia in animal models of persistent inflammatory hyperalgesia, whereas the corresponding B2 receptor antagonists were ineffective or weakly active in these models (Perkins et al., Eaia (1993) 52:191-197). One limitation of the bradykinin antagonist peptides known to date is the necessity for parenteral administration. Due to the peptidic nature of the compounds, they are unlikely to be orally active. Further, peptides in general tend to have a relatively short duration of action as a consequence of their rapid metabolic degradation. As a result, non-peptide or pseudopeptide bradykinin receptor antagonists that lack the limitations of a peptide offer meaningful therapeutic advantages. DISCLOSURE OF TNVENTTON The present invention resides in the discovery that the novel pseudopeptide compounds identified below, are potent bradykinin receptor antagonists. The compounds are useful in the treatment of various diseases including inflammatory disorders, asthma, septic shock, and burn pain. Included in the invention are pharmaceutical compositions containing the inventive compounds and methods of using the compounds as bradykinin receptor antagonists. The compounds of the invention which interact with the B2 receptors have the formula: X-Y-Z wherein, X is a moiety having a net positive charge; Y is a hydrophobic organic moiety having a nitrogen atom at the X- Y junction, a carbonyl group at the Y-Z junction, an atomic volume in the range of 135A3 to 300A3, and an end-to-end distance between the flanking nitrogen and carbonyl atoms of about 5.θA ± 1.5 A; Z is an arrangement of atoms which inherently adopt a beta turn conformation and has a positive charge near the distal end. Preferred compounds of the formula X-Y-Z are those which have a binding affinity constant (Kø of less than 500 nm, more preferably less than 100 nm, for membrane bound human bradykinin B2 receptor, using the assay procedure described herein. In another embodiment, the invention relates to pseudopeptide compounds capable of binding to the bradykinin Bi receptor. These compounds, because of their ability to block B 1 receptor mediated hyperalgesia, are useful in the treatment of conditions associated with chronic inflammation. In particular, these compounds have the same structure as that set forth for the B2 receptor antagonists described above with the exception that they lack the posively charged moiety at the distal end of the Z group. The compounds of the invention have numerous advantages over conventional peptide bradykinin agonists. The most salient advantage is the elimination of many (or all) of the amide bonds from the bradykinin-like structure. Reduction or elimination of the peptide nature of the compounds leads to compounds having an increased duration of action. The compounds of the invention are longer lasting and have a wider range of administration modes than conventional peptide bradykinin antagonists (e.g., do not necessarily require 0 parenteral administration, may possibly be orally active). The compounds of the invention are also less expensive to prepare than peptide bradykinin antagonists. Another embodiment of the invention involves a pharmaceutical composition useful as a bradykinin receptor antagonist comprising a pharmaceutical carrier and an effective amount of the compound of the invention. 5 The invention also involves a process for antagonizing bradykinin receptor activity in mammals which comprises administering to a subject an effective amount of the novel compound to antagonize bradykinin receptor activity. A further embodiment involves a pharmaceutical preparation for treating local pain and inflammation from burns, wounds, cuts, rashes and other such 0 trauma, and pathological conditions caused by the production of bradykinin or related kinins by an animal which comprises administering an effective amount of the novel compound sufficient to antagonize bradykinin with a suitable pharmaceutical carrier. Another aspect of this invention involves a process for treating local pain and inflammation which comprises administering an effective 5 amount of the pharmaceutical preparation to an animal in need thereof. MODES FOR CARRYING OUT THE INVENTION The compounds of the invention which are capable of specifically competing with native bradykinin for binding to the bradykinin B2 receptor have _ Λ the following structure: X-Y-Z wherein, X is a moiety having a net positive charge selected from the group comprising a positively charged amino acid and an organic group; ,.,. Y is a hydrophobic organic moiety having the following characteristics: a. a nitrogen atom at the X-Y junction; b. a carbonyl group at the Y-Z junction; c. the hydrophobic organic moiety between the nitrogen atom and the carbonyl group is selected from the group consisting of a carbocyclic, heterocyclic and a linear organic moiety; d. an atomic volume in the range of 135 A3 to 300 A3; e. an allowed conformation such that an end-to-end distance between the flanking nitrogen and carbonyl atoms is about 5.0 A ± 1.5 A; f. with the proviso that Y cannot consist of naturally occuring amino acids; Z is an arrangement of atoms which inherently adopt a beta turn conformation and has a positive charge near the distal end; and pharmaceutically acceptable salts thereof. The compounds of the invention which are capable of specifically competing with des Arg^-bradykinin for binding to the bradykinin B i receptor have the following structure: X-Y-Z' wherein, X and Y are as defined above and Z' is an arrangement of atoms which inherently adopt a beta turn conformation and which lacks a positive charge near the distal end; and pharmaceutically acceptable salts thereof. A. Preferred X Moieties X is a moiety having a net positive charge. Preferably X comprises a positively charged amino acid or an organic group. Alternatively, X can comprise both a positively charged amino acid and an organic group. When X comprises a positively charged amino acid, it preferably is a mono-, di- or tri-peptide independently selected from the group consisting of the L- and D- isomers of Arg, Gin, Asn, Lys, Sar, N-e-acetyl-Lys, N^-p-tosyl-Arg-NC-nitro-Arg, acetyl- Arg, and citrulline. Most preferably X is D-Arg-Arg. When the group Y is as described in Formula 2, below, then X may also preferably by a tripeptide (I-II-m) in which amino acids I and π are independently selected from the group consisting of the L- and D-isomers of Arg, Gin, Asn, Lys, Sar, N-ε-acetyl-Lys, NC-p-tosyl-ArgNC-nitro-Arg, acetyl-Arg and citruline ,and amino acid HI is selected from the group consisting of Pro, 4Hyp and Oic. A preferred tripeptide representing X, when Y is described in Formula 2, is D-Arg-Arg-Pro. When X is an organic moiety it can include, but is not limited to, triazole, 8-guanidinooctanoic acids, 10-aminodecanoic acids and N-[(5-guanidino)pentyl]- Arg. In these organic moieties the alkyl bridge may be substituted, branched, straight, cyclic, saturated or unsaturated. B. Preferred Y Moieties Y is a hydrophobic organic moiety having the following characteristics: a. a nitrogen atom at the X-Y junction; b. a carbonyl group at the Y-Z junction; 5 c. the hydrophobic organic moiety between the nitrogen atom and the carbonyl group is selected from the group consisting of a carbocyclic, heterocyclic and a linear organic moiety; d. an atomic volume in the range of 135 A3 to 300 A3; e. an allowed conformation such that an end-to-end distance between the ϋ flanking nitrogen and carbonyl atoms is about 5.0 A ± 1.5 A; f . with the proviso that Y cannot consist of naturally occuring amino acids. The hydrophobic organic moiety Y must satisfy at least a two part criterion. The first criteria is related to a conformationally allowed end-to-end 15 length of the isolated moiety and the second criteria is related to atomic volume. When considered in isolation from the complete molecule (X-Y-Z), Y must be able to adopt an N-terminal to C-terminal distance of about 5.θA ± 1.5 A. The end-to-end distance between the flanking nitrogen and carbonyl atoms of about 5.θA ± 1.5 A is achieved via either inherent flexibility or inherent rigidity. 2 ^ This geometric constraint might be a function of some inherent structural rigidity imparted, for example, by unsaturation or cyclization. Alternatively, this preferred geometry might be attainable geometry in which the fragment Y is able to satisfy the end-to-end distance via inherently flexible conformational changes which would be allowed on the basis of a Boltzmann distribution of * conformational states at room temperature. Further, an acceptable Y has an atomic volume of not less than 135 A3 and not greater than 300A3 as determined by the numerical integration method of Pearlstein, R.A. (Thesis, Department of Macromolecular Science, Case Western Reserve University, Cleveland, OH, 1983). Preferably, Y is selected from the 3^ group consisting of carbocyclic, heterocyclic, polycyclic and linear (saturated and unsaturated) organic molecules. Y cannot comprise a natural amino acid. Y moieties which do not satisfy both parts of the criterion, length and volume, are not acceptable as Y in the compounds of the invention. Several of the hydrophobic spacer groups (Y) specifically defined by this 5 invention include, but are not limited to: 1 ,2-benzo-4-keto-3,8-diazaspiro[4.5]decan-3-alkanoic acids, l,2,3,4-tetrohydro-9H-pyrido[3,4-b]indol-9-alkanoic acids (β-carboline- alkanoic acids), l,2,3,4-tetrahydro-5H-pyrido[4,3-b]indol-5-alkanoic acids (γ-carboline- alkanoic acids), amino-2-quinolinon-l-alkanoic acids, amino-6-(5H)phenanthridinon-5- alkanoic acids, l-(4-piperidinyl)-2-benzimidazolon-3-alkanoic acids, N- (aminophenyl)-N-benzoylglycine-N-alkanoic acids, co-aminoalkanoic acids, substituted ω-aminoalkanoic acids, olefinic ω-aminoalkenoic acids, and 4-keto- 1 ,3,8-triazaspiro[4.5]decan-3-alkanoic acids. In various embodiments of the invention, the Y group can have any of the formulae selected from Formula 1, Formula 2 and Formula 3 below: Formula 1 wherein R is selected from the group consisting of a substituted or unsubstituted aryl group, a straight, branched, saturated or unsaturated lower alkyl chain of 1 to 6 carbon atoms, and a cycloalkyl or cycloalkylmethyl in which the cycloalkyl ring comprises 3 to 6 carbon atoms; and R 2 is a saturated or unsaturated alkylene brigde consisting of 1 to 8 carbon atoms optionally substituted with a benzyl or napthyl group or in which one of the carbon atoms of the bridge is disubstituted to form a cycloalkyl ring consisting of 3 to 6 carbon atoms. In Formula 1, R^ is preferably selected from the group consisting of a cyclohexyl, phenyl, naphthyl, benzyl or naphthylmethyl group optionally substituted with a Ci to C2 lower alkyl group, a Ci to C4 alkoxy group or a halogen. More preferably, R is selected from the group consisting of phenyl, 4-methylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, benzyl, 1 -naphthyl, 2-naphthyl, cyclohexyl, cyclohexylmethyl, n-propyl, n-pentyl and neopentyl. Most preferably, R is selected from the group consisting of phenyl, 4-methylphenyl, cyclohexyl, cyclohexylmethyl and n-propyl. In Formula 1, R 2 is preferably a saturated or unsaturated alkylene bridging group consisting of 1 to 8 carbon atoms optionally substituted with a benzyl or naphthyl group. More preferably, R 2 is a saturated or unsaturated alkylene bridging group consisting of 1 to 4 carbon atoms. Most preferably, R 2 is (CH2) X wherein x is an integer from 1 to 4. Formula 2 R 7 O -R 3 3 -R 4 4 -(CH 2 ) m -R 5 5 -(CH 2 ) n -R 6 6 -(CH 2 ) 0 - 'CH- "C- wherein R 3 is selected from the group consisting of 2-pyrrolidinyl, Pro, 4Hyp, Oic, dehydroPro, Tic, Aoc, L-azetidine-2-carboxylic acid, Eac, Gly, Thz, ιυ and Aib; R 4 is selected from the group consisting of a direct bond and an imino (-NH-) group; R 5 and R 6 are independently selected from the group consisting of a direct bond, a C3 to Cs carbocycle and a C2 to s monoolefin or a C4 to Cis polyolefin containing 2 to 5 double bonds which may optionaly be incorporated into a cyclic system; R 7 is selected from the group consisting of hydrogen, hydroxymethyl, to alkyl, benzyl, thiophenylmethyl and furanylmethyl; and m, n, and o are independently an integer from 0 through 12, with the proviso that their total does not exceed 12. In Formula 2, R 3 is preferably selected from the group consisting of 2-pyrrolidinyl, Pro, 4Hyp and Oic. More preferably, R 3 is selected from 10 2-pyrrolidinyl and Oic. Preferred Y groups of Formula 2 include those wherein R 5 is selected from the group consisting of a direct bond, a C3 to Cs carbocycle, and a C2 to Cis monolefin or C4 to Cis polyolefin containing 2 to 5 double bonds which may optionally be incorporated into a cyclic system; R 6 is selected from the group consisting of a direct bond and a C2 to Ci8 monoolefin; R 7 is selected from the group consisting of hydrogen and benzyl; and m, n, and 0 are independently an integer from 0 to 6. 30 35 Preferred cyclic systems incorporated into R 5 and R 6 include: Preferred Y groups of Formula 2 in which R is a direct bond include, but are not limited to: 4-amino-2-butenoyl; o 3-[2-(aminomethyl)phenyl]-2-propenoyl; 3-[2-(aminomethyl)phenyl]-2-propanoyl; 3-[3-(aminomethyl)phenyl]-2-propenoyl; 3-[3-(aminomethyl)phenyl]-2-propanoyl; 4-[2-(aminomethyl)phenyl]-3-butenoyl; 3-[2-(aminoethyl)phenyl]-2-propenoyl; 6-amino-4,5-( 1 ,2-cyclohexyl)-2-hexenoyl; 30 35 Formula 3 O II -NH-(CH 2 ) m -R 5 -(CH 2 ) n -R 6 -(CH 2 ) 0 -C- wherein R 5 , R 6 , m, n, and o are as previously defined for Formula 2. Exemplary of other hydrophobic spacer groups (Y), which satisfy the criteria set forth above for Y are the following, which are intended to be illustrative only and not to limit the scope of the invention: In the above formulae, z is an integer from 1 to 3. Examples of routes of synthesis of protected hydrophobic spacer moieties (Y) described above, which are suitable for incorporation into the pseudopeptide or non-peptide bradykinin antagonists of the invention by known coupling techniques, are shown below in Schemes I -XIV . For incorporation of these aminoacyl groups into the bradykinin antagonists of the invention it is necessary to protect the amino functionality with, for example, Boc (tert-butoxycarbonyl) or FMOC (9-fluorenylmethoxycarbonyl) groups. The required N-protected amino acids are generally derived by a sequence that involves addition of an amine-protecting group to an ester of the amino acid spacer group, followed by hydrolysis of the ester. In most instances, requisite esters are obtained by alkylation of an amide or amide-like precursor (see Schemes I-V) with an alkyl haloalkanoate. Following acylation with a suitable protecting group, the protected amino-ester is hydrolyzed to the protected amino acid. The amide or amide-like starting materials have been described previously, are commercially available or are prepared by conventional chemical synthetic procedures that are well known to those skilled in the art. As shown in Scheme I, bis-alkylation of oxindole with bis- 10 chloroethylmethylamine affords a spiro-piperidine which is alkylated with methyl bromoacetate. Sequential N-demethylation, Boc protection and ester hydrolysis of the dervived acetate affords N-Boc-l,2-benzo-4-keto-3,8-diazaspiro[4.5]decan- 3-acetic acid. 15 Scheme I CCHH 33 NN((CCHH 22 CCHH 22 CC11)) 22 Boc-protected l,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-9-alkanoic acids are derived from l,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole ( 1,2,3 ,4-tetrahydro- β-carboline (Aldrich Chemical Company, Milwaukee, WI) by similar Boc- 3 Q protection, followed by alkylation with ethyl bromoacetate or ethyl 3-bromopropionate and ester hydrolysis. As illustrated in Scheme π, N-Boc-1, 2,3,4- tetrahydro-5H-pyrido [4,3-b]indol-9-acetic acid is derived from N-carbethoxy 4-piperidone and phenylhydrazine condensation in a Fischer indole synthesis, followed by gc carbethoxyalkylation, Boc protection and ester hydrolysis. Scheme II Boc 7-(9-FMOC-amino)-4-methyl-2-quinolion-l -acetic acid is derived from 7-amino-4-methyl-2-quinolinone (Chilin et al., J. Org. Chem. (1991) 56:980-983) in a related alkylation, N-protection, hydrolysis sequence as illustrated in Scheme HI. Scheme III Boc-protected 3-amino-6(5H)phenanthridinon-6-acetic acid is prepared from 3-nitro-6(5H)phenanthridinon (Rare Chemical Collection, Aldrich Chemical Company, Milwaukee, WI) by methyl bromoacetate alkylation, nitro group reduction, amine protection and ester hydrolysis as outlined in Scheme IV. Scheme IV As shown in Scheme V, a similar procedure is employed to prepare Boc- protected N-aminophenyl-N-benzoylglycine-N-acetic acid. Scheme V Boc-protected l-(4-piperidinyl)-2-benzimidazolinon-3-acetic acid is derived from l-(4-piperdinyl)-2-benzimidazolinone (Aldrich Chemical Company, Milwaukee, WI) by alkylation with ethyl iodoacetate followed by Boc acylation and ester hydrolysis. Boc-protected ω-alkanoic acids, as well as their substituted and olefinic derivatives, and substituted 4-keto-l,3,8-triazaspiro[4.5]decan-3-alkanoic acids are obtained as described in co-owned, copending U.S. patent applications. Scheme VI Preparation of protected hydrophobic spacer group (Y) of Formula 1: 1. Pd/C, H 2 HCONH, 1 (BocJ j O R' I wherein R 1 and R 2 are as defined for Formula 1; R 8 is CH3, C2H 5 or benzyl (Bn). Preparation of an N-Boc protected substituted ω-aminoalkanoic acid for introduction of a hydrophobic spacer (Y) of Formula 2 (wherein R 4 is an imino group) into the pseudopeptides of this invention is illustrated by the synthesis of N-Boc-5-amino-2-benzyl-3-pentenoic acid (10) as outlined in Scheme VΗ (in which B n represents benzyl). Scheme VII Ph,P COOMe -|-Siθ OMs COOMe NaN-, Accordingly, diethyl benzylmalonate Q) is reduced with lithium aluminum hydride to afford 2-benzyl-l,3-propanediol (2) which is treated with an equimolar amount of tert-butyldimethylsilyl chloride to give the monoprotected alcohol 2- Swern (dimethylsulfoxide/oxalyl chloride) oxidation of 2. provides the aldehyde 4_which is condensed with the Wittig reagent methyl (triphenylphosphonylidene) acetate to give £. Conversion of the alcohol £ to the mesylate 2 followed by azide displacement affords & which is sequentially reduced and Boc protected to give £. Jones oxidation of the tert- butyldimethylsilyl ether 9_ provides the Boc protected subsituted CD-aminoalkenoic acid lQ. By alteration of the benzyl substituent of the malonate starting material the group R 7 of the general structure of Formula 2 may be varied. Modification of the reagent employed for Wittig condensation with the aldehyde, e.g., 4, enables variation of R 5 , R 6 , m, n, and o of the aminoalkenoyl group of Formula 2 that is introduced into the pseudopeptides of this invention. The general method utilized to prepare mono- and polyolefinic substituted ω-(2-pyrrolidinyl)alkenoic acids for introduction of the fragments wherein R 3 is 2-pyrrolidinyl and the remainder of the Y group is an alkenoyl group into the pseudopeptides of this invention is illustrated by the synthesis of (2S-2S-benzyl- 7-(2S-pyrrolidinyl)-6-heptenoic acid Q8) as outlined in Scheme VIE. Substituted ω-(2-pyrrolidinyl)alkanoic acids 19 are obtained by catalytic hydrogenation of olefinic precursors as shown in Scheme VHI (in which B n represents a benzyl group). Scheme VIII .CH, Ph,P=' " OH COOH CON CHO N N I I OCH, I Boc Boc Boc ii 12 12 14 15. l≤ 12 1£ 12 The initial step in this sequence involves conversion of Boc-protected L(S)-proline (11) to 2-pyrrolidinecarboxaldehyde (13) via Boc-protected N-methoxyl-N-methyl-L-prolinamide (12) according to previously described nethodology [Fehrentz, J.A. and Castro, B., Synthesis (1983) 676-678; Hocart, S.J., et al., J. Med. Chem. (1988) 21:1920-1824; Νahm, S. and Weinreb, S.M., Tet. Lettr. (1981) 22:8315-3818]. Condensation of H with the Wittig reagent derived from 6-bromohexanoic acid and triphenylphosphine according to the general method of Corey, H.S., Jr. et al. fJ. Am. Chem. Soc. (1964) £& 1884] produced Boc-protected 7-(2-pyrrolidinyl)-6-heptenoic acid (14) which is stereoselectively benzylated via the chiral amide 15 to produce Jjji according to a previously described general procedure [Mavunkel, B.J., et al., Tet. Lettr. (1993) 14:22251. Alcoholysis of the amide with benzyl alcohol gives the ester Y∑ which is hydrolyzed to 18 according to the procedure of Evans, D.R., et al. 17. Org. Chem. (1985) 5JM930-1835]. Hydrogenation of Jj£ results in the saturated benzyl-substituted ω-(2-pyrrolidinyl)alkanoic acid 12- Preparation of hydrophobic spacer groups (Y) of Formula 3 for incorporation into the bradykinin antagonists of the invention is illustrated by Schemes DC to XIV. Scheme DC N-Boc-protected 3-[2-(aminomethyl)phenyl]-2-propenoic acid (Compound D and N-Boc-protected 3-[2(aminomethyl)phenyl]-2-propanoic acid (Compound 2) were prepared via the following scheme: Boc 1 H > Pd-C Scheme X N-Boc-protected 3-[3-(aminomethyl)phenyl]-2-propenoic acid (Compound 2) and 3-[3-(aminomethyl)phenyl]-2-propanoic acid (Compound 4) were prepared by the following scheme: 11 ♦' H 2 , Pd-C 14 Scheme XI N-Boc-protected 4-[2-(aminomethyl)phenyl]-3-butenoic acid (Compound 5) was prepared via the following scheme: DEAL O AcCl P NHBoc - y. 11 Scheme XII N-Boc protected 2-[(2-aminoethyl)phenyl] propenoic acid (Compound 22) was prepared via the following scheme: Scheme Xm N-Boc-protected 4-amino-2-butenoic acid (Compound 27) was prepared via the sequence outlined in the following scheme: BocHN COOH BocHN 24 21 Ph 3 P=CHCOOMe LiOH/MeOH/H 2 0 BocHN' "**" " COOMe BocHN' ^*" " COOH CH 3 CN 26 22 Scheme XIV N-Boc-protected 6 amino-4,5-(l,2-cyclohexyl)-2-hexanoic acid (Compound 21) and its isomer (Compound 22) were prepared via the sequence outlined in the following scheme: l. NRjOAc NaCNBH 3 MeOH COOMe LiOH/MeOH/H2θ C( 2. Boc 2 0 07 NHBoc o y NHBoc 20. 21 21 C. Preferred Z and Z' Moieties Z is an arrangement of atoms which inherently adopt a beta turn conformation and has a positive charge near the distal end. Z can be a group of naturally-occurring or non-naturally-occurring amino acids or it can be an non-amino acid organic moiety. When Z is an amino acid-containing moiety it preferably has the formula E-F-G-H-Cn, wherein E is a direct bond or selected from the group consisting of Ser, Thr, Gly, N-BnGly, Val, Ala, Cys and Tyr; F is selected from the group consisting of a D-aromatic amino acid and a D-Hype; G is selected from the group consisting of Oic, Aoc, Thz, octahydro-lH- isoindole-1-carboxylic acid, pipecolinic acid, Pro, 4Ηyp, azetidine- 2-carboxylic acid, Aib, Leu, He, Val, an aromatic amino acid that is Phe, Thi, Tic, indoline-2-carboxylic acid, homoPhe, Trp, Tyr, Nal, and Hype; H is selected from the group consisting of Arg, Orn, Asn, Gin, and Lys; Cn is a hydroxyl group or a C-terminal extension selected from the group consisting of carboxamido, alkoxy, an acidic, basic or natural aliphatic, aromatic, or cyclic amino acid residue of the D- or L- configuration. Most preferably E is a direct bond or is selected from the group consisting of Ser, Gly and Val; F is selected from the group consisting of D-Phe, D-Tic and a D-Hype; G is selected from the group consisting of Phe, Oic, Aoc, Tic and a Hype; H is Arg; and Cn is selected from the group consisting of hydroxyl group, an amide group and an alkoxy group. Preferred Z' moieties correspond to the preferred Z moieties with the exception that they lack the positively charged group, usually a nitrogen- containing moiety, at the distal end. When Z' is an amino acid-containing moiety, it preferably has the formula E-F-G-Cn, wherein E, F and G and Cn are as described above. A number of non-amino acid-containing beta turn mimetics are known. These molecules may be utilized in place of -F-G- for incorporation of the group into the compounds of this invention. In order to facilitate its incorporation into the bradykinin antagonist compound of this invention by techniques described herein, the beta turn mimetic should have an amino terminus and a carboxyl terminus. Some of such beta turn mimics are illustrated by, but are not limited to, compounds that have been described previously. These include moieties with a phenoxathiin ring system, e.g. 1 (M. Feigel, J Am. Chem. Soc. (1986) 108:181- 182), bicyclic dipeptides, e.g. (3S, 6S, 9R)-2-oxo-3-amino-7-thia-l-azabicyclo [4.3.0]nonane-9-carboxylic acid 2 (K. Sato and U. Nagui, J. Chem. Soc. Perkin Trans. (1986) 1231-1234) and some of its derivatives (U. Nagui and K Sato. Tet. Lettr (1985) 26:647-650; U. Nagui and K. Sato: In Deber, CM.; Hruby, V.J. and Kopple, K.D. (Eds.) Peptides: Structure and Function. Proceedings of the 9th American Peptide Svmposition. Pierce Chemical Co., Rockford, IL, 1985, p. 465), 9-(e.g. 3, G.L. Olson et al. J. Am. Chem. Soc. (1990) 112:323-333), 10-(e.g. 4, D.S. Kemp and W.E. Stites, Tet Lettr (1988) 4Q:5057-5060) and Soc. (1988) IH): 1638- 1639) membered moderately constrained rings, a piperdine ring, e.g. 3-amino-2-piperdine-6-carboxylic acid 6 (D.S. Kemp and P.E.McNamara, J. Org. Chem. (1984) 42:2286-2288; D.S. Kemp and P.E. McNamara, J. Ore. Chem. (1985) 5Q:5834-5838; D.S. Kemp and E.T. Sun, Tet Lettr. (1982) 22:3759-3760; D.S. Kemp and P. McNamara TgLisEk (1982) 22:3761-3764), a spirocyclic ring system, e.g. l,δ-diaza-7-oxospiro[5.4]decane-δ-acetic acid 7 (M.J. Cenin, et al., J. Org. Chem. (1993) 51:860-866) and related compounds (M.G. Hinds, et al., J. Med. Chem. (1991) 24:1777-1789), and 5H-6-oxo- 2,3,4,4a,7,7a-hexahydropyrano[2,3-b7pyrroles, e.g. 8 (J.L. Krstenansky, et al., Biochem. Biophvs. Res. Commun. (1982) 109:1368-1374; J.L. Krstenansky et al., J. Heterocyclic Chem. (1992) 22707-711). The following terms used herein in the specification and claims are further defined: olefinic aminoalkenoyl" is a carbon chain of from 2 to 18 carbons containing at least one double bond, wherein 2 to 4 carbons may be optionally incorporated into a cyclic structure, having an amino acid linkage (i.e., an N-terminal amino group and a C-terminal carbonyl group). The alkenyl portion of the olefinic aminoalkenoyl is preferrably a hydrocarbon chain, but may also include carbon replacements, such as by nitrogen. "Hype" is defined herein as having the following structure: R- X i o wherein R is selected from the group consisting of H, Ci-Cβ alkyl, substituted C1-C6 alkyl, C2-C8 alkenyl, C2-C8 cycloalkyl, C3-C8 cycloalkyl substituted C1-C6 alkyl, an aryl group, a substituted aryl group wherein the substituent is C1-C4 alkyl, C1-C4 alkoxy, halogen or trifluoromethyl, an arylalkyl group, and a group of the formula R 1 NHC(O) where R 1 is Ci- ; alkyl or aryl, and where X is either SOn or oxygen, and n=0, 1, or 2; "carbocyle" and "cycloalkyl" are interchangeably defined herein as a saturated cyclic hydrocarbon structure, such as cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl and cycloheptyl, this definition includes mono- and polycyclic structures; "olefin" and "cycloalkenyl" are defined herein as a cyclic hydrocarbon structure containing at least one double bond and includes substituted aryl groups, such as 1,2-, 1,3- and l,4-phenylene,benzyl, phenyl, cyclohexenyl, cyclohexadienyl, cyclopentadienyl. Also included in this definition are multiple ring structures, such as naphthyl; "pseudopeptide" is an entity which is partially amino acid (peptidic) in nature and partially organic chemical in nature. A minimum of two peptide bonds re eliminated and replaced by organic molecules having the ability to retain the functionality of amino acids they replace in the pseudopeptides of the present invention; "alkenyl" and "olefin" are interchangeably defined herein as a hydrocarbon structure containing at least one double bond, suitable alkenyls can also be hydrocarbon structure containing multiple double bonds, the double bonds can optionally be incorporated into a ring structure, such as a cycloalkenyl; "amino acid linkage" is exemplified by a moiety having a N-terminal amino group and a C-terminal carbonyl group; "alkyl" is a paraffinic hydrocarbon group which may be derived from an alkane by dropping one hydrogen from the formula, such as methyl, ethyl, propyl, isopropyl, butyl, and so forth; "substituted C1-C6 alkyl" is a branched alkyl, such as methylbutyl; "aryl" is an aromatic ring compound such as phenyl, naphthyl; "substituted aryl" is a substituted aromatic ring including, but not limited to, nitro substitution, or halogen substitution; and "aralkyl" is a aryl being attached through an alkyl chain, straight or branched, containing from one through six carbons, such as a phenylpropyl group. A "direct bond" is a bond which replaces a particular amino acid compound between adjacent amino acids and which amino acid may also be indicated to be absent by the term "null". The phrase "a suitable amine protecting group" is a group, such as Boc (t-butyloxycarbonyl-) which protects the amine moiety from reaction and which can be removed under mild conditions so as not to affect the rest of the molecule. Definitions of the amino acid abbreviations used herein are as follows: "amino acid" is the basic structural unit of proteins, consisting of an amino group, a carboxyl group, a hydrogen atom and a distinctive R group (side chain) bonded to a carbon atom. There are 20 naturally occuring amino acids which have the following abbreviations: Ala is alanine; Arg is arginine; Asn is asparagine; Asp is aspartic acid; Cys is cysteine; Gin is glutamine; Glu is glutamic acid; Gly is glycine; His is histidine; De is isoleucine; Leu is leucine; Lys is lysine; Met is methionine; Phe is phenylalanine; Pro is proline; Ser is serine; Thr is threonine; Trp is tryptophan; Tyr is tyrosine; and Val is valine. There are also numerous well-known non-naturally occuring amino acids which include, but are not limited to the following: Aib is 2-aminoisobytyric acid; Aoc is (S,S,S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid; Eac is e-aminocaproic acid; Nal is beta-2-naphthylalanine; Orn is ornithine; dehydroPro is 3,4-dehydroproline, homoPhe is homophenylalanine; 4Hyp is 4-hydroxyproline; Sar is sarcosine; Thi is beta-2-thienylalanine; Thz is thiazolidine-4-carboxylic acid; phenylGly is 2-phenylglycine; Tic is tetrahydroisquinoline-3-carboxylic acid; Oic is (2S, 3aS, 7aS)-octahydro-lH-indole-2-carboxylic acid; prenyl is a 3-methyl-2-butenyl radical. "aromatic amino acid" is a naturally occurring or non-naturally occurring amino acid having one or more unsaturated carbon rings and includes, but is not limited to, Phe, Tic, Thi, n-benzyl Gly, homoPhe, Tyr, Trp, indoline-s-carboxylic acid, Nal. D-Ηype (trans-propyl) is 4S-D-prolyl propyl ether and represents: D-Hype (trans-thiophenyl) is 4S- D-prolyl phenyl thioether, also known as D-4-hydroxyproline trans phenylthioether also known as D-Hyp S(trans-phenyl) and represents: D-Hype (trans-phenylpropyl) represents D-Hype (trans-2-methylbutyl) represents: D-Hype (trans-ethyl) represents: D-Hype (trans-methyl) represents: Aoc can be prepared by the method of V. Teetz, R. Geiger and H. Gaul, Tetrahedron Lett. (1984):4479. Tic is commercially available from Bachem Biosciences or can be prepared by the method of K. Hayashi, Y. Ozaki, K. Nunami and N. Yoneda, Chem. Pharm. Bull. (1983) 21:312. All amino acids residues, except Gly and Ser, described in the specification are preferably of the L-configuration unless otherwise specified. It would be recognized, however, that the 7 position amino acids and derivatives must always be the D-configuration whereas the amino acids and derivatives of position 8 may be either in the D- or L- configuation. The hydroxyproline ethers at position 7 are preferably in a trans configuration, whereas the hydroxyproline ethers at position 8 can be in either the cis or trans configuration. The symbols and abbreviations used for amino acids, their derivatives and protecting groups, and peptides and their salts are those customarily used in peptide chemistry. (See Biochem. J. (1972) 126:773), which Journal reference is hereby incorporated by reference. The synthesis of the peptides of this invention including derivation, activation, and coupling of protected amino acid residues, and their purification, and the analytical methods for determining identity and purity are included in the general body of knowledge of peptide chemistry, as described in Houben Weyl Methoden der Organischen Chemie ( 1974) Vol. 16, parts I & II for solution-phase synthesis, and in Solid Phase Peptide Synthesis. (1984), by Stewart and Young for synthesis by the solid-phase method of Merrifield. Any chemist skilled in the art of peptide synthesis can synthesize the peptides of this invention by standard solution methods or by manual or automated solid phase methods. The appropriate hydroxyproline substituents used at position 7 or 8 are prepared by the process described in PCT publications WO 92/18155 and WO 92/18156 which are herein incorporated by reference. The starting materials are commercially available and can be prepared by known procedures. Both the cis and trans stereoisomers can be prepared by these means and are within the scope of the present invention. INDUSTRIAL APPLICABILITY Therapeutic applications of the novel bradykinin antagonists include not only treatment for the production of bradykinin or related kinins by the animal but also the injection of bradykinin related peptides into an animal as a result of bites and stings. Topical application alone or in combination with subcutaneous utilization of the bradykinin antagonists of the invention can be employed to treat the effects of bradykinin-related peptides causing pain, inflammation and swelling. The therapeutic use of bradykinin antagonists of this invention for other traumatic inflammatory or pathological conditions which are known to be mediated by bradykinin or exacerbated by an overproduction of bradykinin can also be achieved. These conditions include inflammatory disorders such as shock, systemic inflammatory response syndrome, pancreatitits, and angioedema, arthritis and inflammatory bowel disease, systemic treatment of pain and inflammation, local trauma such as wounds, burns, rashes, airway disorders such as asthma, rhinitis and allergies, and nervous system diseases such as spinal cord injury, stroke, hemorrhage, trauma, tumors, access and encephalitis. The compounds of the invention which are Bi receptor antagonists are useful, alone or in combination with the B2 receptor antagonists, for the treatment of conditions associated with persistent inflammatory hyperalgesia, e.g. rheumatoid arthritis. In parenteral administration of the novel compounds and compositions of the invention the compounds may be formulated in aqueous injection solutions which may contain antioxidants, buffers, bacteriostats, etc. Extemporaneous injection solutions may be prepared from sterile pills, granules, or tablets which may contain diluents, dispersing and surface active agents, binders and lubricants which materials are all well known to the ordinary skilled artisan. In the case of oral administration, fine powders or granules of the compound may be formulated with diluents and dispersing and surface active agents, and may be prepared in water or in a syrup, in capsules or cachets in the dry state or in a non-aqueous suspension, where a suspending agent may be included. The compounds may also be administered in tablet form along with optional binders and lubricants, or in a suspension in water or syrup or an oil or in a water/oil emulsion and may include flavoring, preserving, suspending, thickening, and emulsifying agents. The granules or tablets for oral adminstration may be coated or other pharmaceutically acceptable agents and formulations may be utilized which are all known to those skilled in the pharmaceutical art. Solid or liquid carriers can also be used. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, and water. Ointments and creams are prepared using various well known hydrophilic and hydrophobic bases. Topical reservoirs are suitably prepared using known polymeric materials such as various acrylic-based polymers selected to provide desired release characteristics. Suppositories are prepared from standard bases such as polyethylene glycol and cocoa butter. The method of treatment according to this invention comprises administering internally or topically to a subject an effective amount of the active compound. Doses of active compounds in the inventive method and pharmaceutical compositions containing same are an efficacious, nontoxic quantity selected from the range of 0.01 to 500 mg/kg of active compound, preferably 0.1 to 50 mg/kg. Persons skilled in the art using routine clinical testing are able to determine optimum doses for the particular ailment being treated. The desired dose is administered to a subject from 1 to 6 or more times daily, intraveneously, orally, rectally, parenterally, topically, or by inhalation. The efficacy of the inventive compounds of this invention as bradykinin receptor antagonists can be determined using the bradykinin binding and tissue assays described herein. The results of these assays demonstrate that the novel compounds are potent, selective bradykinin receptor antagonists. The following examples are illustrative of preferred embodiments of methods of preparation and compounds of the invention and are not to be construed as limiting the invention thereto. The preparation of various specific hydrophobic spacer groups (Y) useful for incorporation into the bradykinin antagonists at the invention are exemplified in the following Examples 1 through 7. Example 1 1.2-Benzo-8-tert-butoxycarbonyl-4-keto-3. 8-diazaspiror4.51decan-3-acetic Acid (1) Preparation of l,2-benzo-4-keto-8-methyl-3,8-diazaspiro[4.5]decane. A stirred solution of 6.65 g (50 mmol) of oxindole in 100 ml of tetrahydrofuran was cooled to -78°C and 250 ml (250 mmol) of a 1M solution of sodium bis(trimethylsilyl)amide in tetrahydrofuran was added dropwise. The mixture was stirred for 30 minutes at -78°C and then 9.63 g (50 mmol) of N-methyl bis(2-chloroethyl)amine hydrochloride was added. After the mixture was stirred at -78°C for 30 minutes, it was allowed to come to ambient temperature and stirring was continued for 18 hours. Water (100 ml) was added and the mixture was extracted three times with ether. The ether extracts were washed with brine, dried (Na2SO4) and concentrated. The resudue was chromatographed (silica gel, eluting with a 5 to 50% gradient of methanol in methylene chloride) to give 7.17 g (66%) of the product as tan crystals, mp 200-205°C (dec). (2) Preparation of methyl l,2-benzo-8-methyl-4-keto-3,8-diazaspiro[4.5] decan-3-acetate. To a stirred suspension of sodium hydride (1.60 g, 40 mmol, 60% dispersion in mineral oil) in 10 ml of dimethylformamide a solution of 7.07 g (32.7 mmol) of l,2-benzo-4-keto-8-methyl-3, 8-diazaspiro[4.5]decane in 50 ml of dimethylformamide was added dropwise. After the mixture was stirred at 0°C for 30 minutes, 3.8 ml of methyl bromoacetate was added and then the mixture was stirred at ambient temperature for 5 hours. The mixture was poured into 500 ml of water and extracted with five portions of ether. The ether extracts were washed with water, then brine, dried (Na2SO4), and concentrated. The residue was chromatographed on a silica gel column, eluting with a 1 to 25% gradient of methanol in methylene chloride to give 7.93 g. (84%) of the product as crystals, mp 100-103°C. (3) Preparation of methyl l,2-benzo-8-tert-butoxycarbonyl-4-keto-3,8- diazaspiro[4.5]decan-3-acetate. A mixture of 3.26 g (11.3 mmol) of methyl l,2-benzo-8-methyl-4-keto- 3, 8-diazaspiro[4.5]decan-3-acetate, 7.5 ml (45.1 mmol) of 2,2,2-trichloroethyl chloroformate and 100 ml of toluene was stirred and refluxed for 18 hours. After being cooled to ambinent temperature, the mixture was diluted with 200 ml of ether, washed with water, dried (NaSO4) and concentrated. The residual oil was dissolved in 30 ml of acetic acid and 10 g of zinc dust was added. After the initial exotherm subsided the reaction mixture was stirred for 1 hour, 50 ml of methylene chloride was added and the mixture was filtered through diatomaceous earth. The filtrate was diluted with 50 ml of water, cooled to 0°C and made alkaline with concentrated aqueous ammonia. The layers were separated and aqueous phase was extracted with methylene chloride. The combined organic solutions were washed with brine, dried (Na2SO4) and concentrated. The residue was dissolved in 50 ml of dioxane. The solution was cooled to 0°C and a solution of di-tert- butyl flicarbonate in 10 ml of dioxane was added. After being stirred at 25°C for 16 hours, the reaction mixture was diluted with 100 ml of ether and 25 ml of water. The layers were separated and the aqueous phase was extracted three times with ether. The combined organic extracts were washed with water and brine, dried (Na2SO4) and concentrated. The residue was chromatographed on a silica gel column eluting with a 0 to 5% gradient of methanol in methylene chloride to give 3.82g (90.3%) of the product as colorless crystals, mp 135-136°C. (4) Preparation of l,2-benzo-8-tert-butoxycarbonyl-4-keto-3,8- diazaspiro[4.5]decan-3-acetic acid. A stirred mixture of 3.76 g (10 mmol) of methyl l,2-benzo-8-te/ - butoxycarbonyl-4-keto-3, δ-diazaspiro[4.5]decan-3-acetate, 4.24 g (40 mmol) of sodium carbonate, 50 ml of methanol and 50 ml of water was heated at reflux for 2 hours. Methanol solvent was distilled off and the resulting solution was diluted with water, cooled to 0°C and acidified with 5N hydrochloric acid. The mixture was extracted five times with ethyl acetate. The extracts were washed with brine, dried (Na2SO4) and concentrated. Recrystallization of the residue from ethanol afforded 2.67 g (74.2%) of the product as crystals, mp 225-228°C (dec). Example 2 5-r2-( r tgrt-Butoxycarbonyl)-1.2.3.4-tetrahvdro-5H-pyridor4.3-b1indole1acetic Acid (1) Preparation of 2-carbethoxy -l,2,3,4-tetrahydro-5H-pyrido[4,3-b] indole. A stirred mixture of 5.2 g (30 mmol) of l-carbethoxy-4-piperidone and 4.34 g (30 mmol) of phenylhydrazine hydrochloride in 9 ml of pyridine under argon was heated in an oil bath at 110-115°C for 18 hours. After being cooled to 25°C, the reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed with water, dried over Na2SO4 and concentrated. Chromatography of the residue on a silica gel column eluting with a 0 to 30% gradient of ethyl acetate in hexane afforded the product (80%) as colorless crystals, mp 126-127°C. (2) Preparation of methyl 5-[2-carbethoxy- 1 ,2,3,4-tetrahydro-5H-pyrido- [4,3-b]indole]acetate. To a stirred suspension of 264 mg (11 mmol) of sodium hydride in 15 ml of dimethylformamide under argon at 5°C was added dropwise a solution of 2.44 g (10 mmol) of 2-carbethoxy-l,2,3,4-tetrahydro-5H-pyrido 4,3-b7indole in 15 ml of dimethylformamide. After the mixture was stirred at 25°C for 30 minutes, 1.53 g (10 mmol) of methyl bromoacetate was added and stirring at 25°C was continued for 24 hours. The reaction mixture was quenched by addition of a 100 ml of a saturated aqueous ammonium chloride solution. The mixture was extracted with ethyl acetate. The extract was washed with water, dried (Na2SO4) and concentrated. Chromatography of the residue on silica gel eluting with a 0 to 30% gradient of ethyl acetate in hexane gave the product (67%) as an oil. (3) Preparation of 5-[2-(tert-butoxycarbonyl)-l,2,3,4-tetrahydro-5H- pyrido[4,3-b]indole]acetic acid. After a mixture of 1.63 g (mmol) of methyl 5-[2-carbethoxy-l,2,3,4- tetrahydro-5H-pyrido[4,3-b]indole]acetate, 5.2 ml of 10% aqueous potassium hydroxide solution and 20 ml of ethanol was stiπ-ed and refluxed for 20 hours, it was cooled to 25°C, the pΗ was adjusted to 9 by addition of IN hydrochloric acid, 1.5 g (6.9 mmol) of di-tert-butyl dicarbonate was added and stirring was continued for an additional 18 hours. The mixture was concentrated to remove ethanol and then it was extracted with ethyl acetate. The extract was dried (Na2SO4) and concentrated. Recrystallization of the residual solid from ethyl acetate gave 1.1 g (64%) of the product as a crystalline solid, mp 202-203°C. Example 3 3-r9-r2- (' t g rt-Bntoxycarbonyl)-1.2.3.4-tetrahvdro-9H-pyridor3.4-b1indole1- propanoic Acid (1) Preparation of 2-(tert-butoxycarbonyl)-l,2,3,4-tetrahydro-9H- pyrido[3,4-b]indole. Di-tert-butyl dicarbonate (14.5 ml, 61.2 mmol) was added to a stirred mixture of 10.1 g (57.5 mmol) of l,2,3,4-tetrahydro-9Η-pyrido[3,4-b/ indole and 6.17 g (58.1 mmol) of sodium carbonate in 120 ml of water and 100 ml of 2-propanol at 25°C. After being stirred at 25°C for 16 hours the mixture was diluted with 100 ml of water and extracted with ethyl acetate. The extracts were washed with brine, dried over sodium sulfate and concentrated. The residual solid was recrystallized from ethyl acetate-hexane to afford 15.36 g (98%) of product as colorless crystals, mp 150-151°C. (2) Preparation of ethyl 3-[9-[2-(tert-butoxycarbonyl)-l,2,3,4-tetrahydro- 9H-pyrido[3,4-b]indole]]propanoate. To a stirred suspension of 0.4 g (13.3 mmol) of an 80% dispersion of sodium hydride in mineral oil in 50 ml of dimethylformamide at 25 °C under argon was added in portions 3.0 g (11.0 mmol) of 2-(tert-butoxycarbonyl)- 1,2,3,4- tetrahydro-9H-pyrido[3,4-b]indole. After the mixture was stirred at 25°C for 30 minutes, 1.6 ml (12.5 mmol) of ethyl 3-bromopropanoate was added. The resulting mixture was stirred at ambient temperature for 16 hours and then it was acidified to Congo red with 5N hydrochloric acid. The mixture was extracted with ethyl acetate. The extracts were washed with brine, dried over sodium sulfate and concentrated to afford the crude product as an oil. (3) Preparation of 3-[9-[2-tert-butoxycarbonyl)- 1 ,2,3,4-tetrahydro-9H- pyrido[3,4-b]indole]]-propanoic acid. Crude ethyl 3-[9-[2-(tert-butoxycarbonyl)-l,2,3,4-tetrohydro-9H-pyrido [3,4-b]]indole]propanoate, prepared as described above, was dissolved in 20 ml of methanol. Sodium hydroxide (3N, 7.5 ml, 2.25 mmol) was added to the methanoic solution and the mixture was stirred at ambient temperature for 16 hours. After the reaction mixture was diluted with water, it was extracted with ether. The aqueous phase was brought to pΗ 4 with 5N hydrochloric acid. The mixture was extracted with ethyl acetate. The extracts were dried over sodium sulfate and concentrated. Residual solid was recrystallized from methanol-ethyl acetate to afford 2.81 g (74%) of the product as fine crystals, mp 152-153.5°C. 9-[2-(tβrt-Butoxycarbonyl)-l,2,3,4-tetrahydro-9H-pyrido[3,4-b[indole] acetic acid was prepared by the method of this example employing ethyl bromoacetate in place of ethyl 3-bromopropanoate to give colorless crystals, mp 80°C (dec). Example 4 7-(9-Fluorenylmethoxycarbonylamino)-4-methyl-2-quinolinon- 1 -acetic Acid (1) Preparation of tert-butyl 7-amino-4-methyl-2-quinolinon-l -acetate. Sodium hydride (450 mg of an 80% dispersion in mineral oil, 15 mmol) was added to a stirred solution of 2.5g (14.3 mmol) of 7-amino-4-methyl-2- quinolone 17. Org. Chem. (1991) 5&980-983] in 40 ml of dimethylformamide under argon at 0°C. After the mixture was stirred for 30 minutes at 25°C, 3 g (15.4 mmol) of tert-butyl bromoacetate was added and stirring was continued for 24 hours. The mixture was poured into 200 ml of water and extracted three times with methylene chloride. The extracts were washed with water, dried (Na2SO4) and concentrated. Recrystallization of the solid residue from ethyl acetate afforded 2.6 g (70%) of the product as crystals, mp 191-193°C. (2) Preparation of tert-butyl 7-(9-fluorenylmethoxycarbonylamino)-4- methyl-2-quinolon- 1 -acetate. 9-Fluorenylmethoxycarbonyl chloride (2 g, 7.74 mmol) was added to a stirred solution of 2 g (6.94 mmol) of tert-butyl-7-amino-4-methyl-2-quinolon-l- acetate in 60 ml of dioxane. After addition of a solution of 0.84 g of sodium bicarbonate in 10 ml of water, the mixture was stirred at 25°C for 3 hours. An additional 0.5 g of 9-fluorenylmethoxycarbonyl chloride was then added and stirring at 25°C was continued for a further 20 hours. The residue was suspended in methylene chloride and filtered. The filtrate was concentrated and the residual solid was recrystallized from methylene chloride-ethyl acetate to give 1.75 g (32.8%) of the product as crystals, mp 223-224°C. (3) Preparation of 7-(9-fluorenylmethoxycarbonylamino)-4-methyl-2- quinolinon-1 -acetic acid. A solution of 2 g (3.92 mmol) of tgrt-butyl 7-(9-fluorenylmethoxy- carbonylamino)-4-methyl-2-quinolon-l -acetate in 60 ml of a 1:1 mixture of trifluoroacetic acid and methylene chloride was stirred at 25 °C for 24 hours. The solvents were removed in vacuo and the residue was recrystallized from chloroform-tetrahydrofuran to yield 75 g (93.5%) of the product as colorless crystals, mp 256-257°C. Example 5 5-( , 3-tgrt-Butoxycarbonylamino-6(5H)phenanthridinone)acetic Acid ( 1 ) Preparation of methyl 5-(3-nitro-6(5H)phenanthridinone)acetate Acetate. To a stirred mixture of 0.3 g (10 mmol) of an 80% dispersion of sodium hydride in mineral oil in 40 ml of dimethylformamide under argon at 0°C was added in portions 2.0 g (8.3 mmol) of 3-nitro-6(5H)phenanthridinone (Rare Chemical Collection, Aldrich Chemical Co., Milwaukee, WI). The mixture was stirred for 30 minutes and then 0.95 ml (10 mmol) of methyl bromoacetate was added and stirring was continued for 20 hours at 25°C. The precipitated product was filtered, washed with water and dried over phosphorus pentoxide in a vacuum desiccator. The solid product was used for further reaction without additional purification. (2) Preparation of methyl 5-(3-tgrt-butoxycarbonylamino- 6(5H)phenanthridinone)acetate A mixture of 1.70 g (5.4 mmol) of crude solid methyl 5-(3-nitro- 6(5H)phenanthridinone)-acetate, prepared as described above, 100 mg of 10% palladium-on-carbon catalyst and 100 ml of ethanol was hydrogenated on a Parr apparatus for 24 hours at 25°C and an initial pressure of 60 psi of hydrogen. The mixture was filtered and the filtrate was concentrated in vacuo. After a mixture of the residue, 2.38 g (10 mmol) of di-tert-butyl dicarbonate, 0.87 of sodium carbonate, 20 ml of 2-propanol and 20 ml of water was stirred at 25 °C for 48 hours, it was adjusted to pΗ 3 with 5N hydrochloric acid and extracted with ethyl acetate. The extract was washed with brine, dried over sodium sulfate and concentrated in vacuo. Column chromatography of the residue (silica gel, ethyl acetate:hexane 1:4) gave 0.45 g (22%) of the product as an oil. (3) Preparation of 5-(3-tert-butoxycarbonylamino-6(5H)phenanthridinone)- acetic acid. A stirred mixture of 0.88 g (2.3 mmol) of methyl 5-(3-tgrt- butoxycarbonylamino-6(5H)phenanthridinone)acetate and 1.0 g (9.4 mmol) of sodium carbonate in 25 ml of methanol and 25 ml of water was heated at reflux for 2 hours. The methanolic solvent was distilled off and then the reaction mixture was adjusted to pΗ3 with 5N hydrochloric acid. The precipitated solid was recrystallized from ethyl acetate-hexane to afford the product as yellow crystals, mp 259-261°C. Example 6 l- ( -t g rt-Butoxycarbonyl-4-piperidinyl)-2-benzimidazolone-3-acetic Acid (1) Preparation of l-(l-tert-Butoxy-4-piperidinyl)-2-benzimidazolone. A mixture of 5.13 g (23.1 mmol) of l-(4-piperidinyl)-2-benzimidazolone, 3.71 g (35 mmol) of sodium carbonate, and 5.5 ml (23.9 mmol) of di-tgrt-butyl dicarbonate in 40 ml of water and 40 ml of 2-propanol was stirred for 16 hours at 25°C. After the mixture was extracted with ethyl acetate, the combined extracts were washed with brine and dried over sodium sulfate. Removal of solvent provided a solid residue that was recrystallized from ethyl acetate to give the product as 6.20 g (92%) of colorless crystals, mp 162-163°C. (2) Preparation of ethyl l-(l-rgrt-Butoxycarbonyl-4-piperdinyl)-2- benzimidazolone-3-acetate. To a stirred suspension of 0.76 g (25.3 mmol) of sodium hydride in 85 ml of tetrahydrofuran under argon was added 5.08 g (16.0 mmol) of l-(l-tert- butoxycarbonyl-4-piperidinyl)-2-benzimidazolone. After the mixture was stirred for 30 minutes at ambient temperature, 2.1 ml (17.4 mmol) of ethyl iodoacetate was added dropwise. The resulting mixture was stirred at reflux temperature for 5 hours and at ambient temperature for 16 hours and then it was concentrated in vacuo. After a solution of the residue in ethyl acetate was washed successively with 0.1N hydrochloric acid, a saturated aqueous solution of sodium bicarbonate and brine, it was dried over sodium sulfate and concentrated. Flash chromatography of the residue (silica gel, ethyl acetate:hexane 1:1) afforded 4.41 g (68%) of the product as an amorphous solid. (3) Preparation of l-(l-tgrt-butoxycarbonyl-4-piperidinyl)-2- benzimidazolone-3-acetic acid. To a stirred solution of 4.20 g (10.4 mmol) of ehtyl l-(l-tgrt- butoxycarbonyl-4-piperidinyl)-2-benzimidazolone-3-acetate in 30 ml of methanol was added dropwise 5.8 ml (17.4 mmol) of 3N sodium hydroxide. After the resulting mixture was stirred for 16 hours at 25°C, water was added and it was extracted with ether. The aqueous phase was acidified to Congo red with 5N hydrochloric acid and the mixture was extracted with ethyl acetate. The extracts were dried over sodium sulfate and concentrated. Recrystallization of the residue from ethyl acetate-methanol gave 3.60 g (92%) of the product as colorless 5 crystals, mp 208-210°C. Example 7 N-Benzoyl-N-G-tgrt-butoxycarbonylaminophenyl)glvcine 25 (1) Preparation of N-benzoyl-3-nitroaniline To a stirred mixture of 5.0 g (35.5 mmol) of 3-nitroaniline and 6.0 ml (452.6 mmol) of triethylamine in 100 ml of methylene chloride at 25°C under argon of benzoyl chloride (5.0 ml, 43.1 mmol) was added dropwise. The resulting 3 Q mixture was stirred at ambient temperature for 16 hours, washed successively with IN hydrochloric acid, saturated aqueous sodium bicarbonate solution and brine, dried (Na2SO4) and concentrated. The residue was recrystallized from methylene chloride to give 6.59 g (77%) of the product as fine crystals, mp 154-155°C. q c (2) Preparation of ethyl N-benzoyl-N-(3-nitrophenyl)glycinate. To a stirred suspension of 0.93 g (31 mmol) of an 80% dispersion of sodium hydride in mineral oil in 75 ml of dimethylformamide at 25 °C, under argon, was added in portions 5.0 g (20.6 mmol) of N-benzoyl-3-nitroaniline. After the mixture was stirred at 25°C for 30 minutes 3.5 ml (31 mmol) of ethyl 2-bromoacetate was added dropwise and stirring was continued for 3 hours. The mixture was quenched with 100 ml of saturated aqueous ammonium chloride solution and extracted 3 times with ethyl acetate. Te extracts were washed with brine, dried (Na2SO4) and concentrated. The residue was chromatographed (silica gel, ethyl acetate:hexane 1:3) to provide 6.35 g (94%) of the product as a light yellow oil. (3) Preparation of ethyl N-benzoyl-N-(3-benzyloxycarbonyl- aminophenyl)glycinate. A mixture of 3.01 g (9.0 mmol) of ethyl N-benzoyl-N-(3-nitroρhenyl)- glycinate, 3.0 ml (12.7 mmol) of di-tgrt-butyl dicarbonate, 0.6 g of 10% palladium-on-carbon catalyst and 100 ml of ethanol was hydrogenated on Parr apparatus at 25 °C and an initial hydrogen pressure of 60 psi for 24 hours. The mixture was filtered and the filtiate was concentrated. Recrystallization of the crystalline residue from ethyl acetate-hexane gave 3.32 g (92%) of the product as white crystals, mp 129.5- 131.5 °C. (4) Preparation of N-benzoyl-N-(3-tert-butoxycarbonylaminophenyl)- glycine. To a stirred solution of 1.3 g (3.3 mmol) of ethyl N-benzoyl N-(3- benzyloxycarbonylaminophenyl)glycinate in 15 ml of methanol was added 2.5 ml (7.5 mmol) of 3N sodium hydroxide. The mixture was stirred at 25 °C for 16 hours, then the methanol solvent was distilled off. The resulting solution was adjusted to pH 3 with 5N hydnochloric acid and the resulting mixture was extracted with ethyl acetate. The extracts were dried (Na2SO4) and concentrated to give a solid residue. Recrystallization of the solid from ethyl acetate-hexane gave 0.9 g (74%) of the product as fine crystals, mp 170- 171.6°C. Example 8 B2 Receptor Antagonist Compound Synthesis The compounds indicated in Table I were synthesized manually using standard solid phase methods and t-Boc chemistry. 1. Boc- Arg(Tos)-PAM resin was used for the purpose. Amino acids and the Boc-protected derivatives of hydrophobic organic moieties (Y) prepared as described in Schemes I through XIV or in Examples 1 through 7 or taught in co-owned, co-pending patent applications were introduced according to the sequence of the compound. 2. Deprotection: The N-terminal t-Boc protection was accomplished by treating the resin-aa/resin-peptide with trifluoroacetic acid/methylene chloride (1:1) for two minutes followed by a similar treatment for 30 minutes. 3. The resin was then washed with methylene chloride and ethanol and neutralized with 10% triethylamine/methylene chloride or 10% dϋsopropylethylamine/methylene chloride. 4. Couplings: All couplings were carried out using the active ester of the amino acid. The active esters of the individual amino acids were generated prior to their introduction into solid phase synthesis. Five equivalents (with respect to loading of the first amino acid on the resin) of the amino acid, hydroxybenzotriazole hydrate and dicyclohexylcarbodiimide or diisopropylcarbodiimide, was incubated for 30 minutes at 0°C in dimethylformamide or methylene chloride/dimethylformamide (1:1) for this purpose. Couplings were followed until no more free amine was detected on the resin using qualitative ninhydrin analysis (Kaiser test). Different unnatural amino acids behave differently during ninhydrin analysis and the color of the resin (after deprotection and coupling) depends on the specific amino acid being used. 5. After coupling, the resin-peptide was washed with dimethyl formamide and methylene chloride before commencement of another cycle of the synthesis. 6. The finished peptidyl-resin was cleaved from the resin using HF (10 mL/g of resin) in the presence of 10% anisole (scavenger). After removal of HF, the peptide resin was washed with ether and the peptide was extracted with 0.1% TFA or 0.2% acetic acid. Lyophilization yielded crude peptide, usually flaky yellow solids were obtained at this stage. 7. The crude peptide was purified using reverse phase high performance liquid chromatography on a Ci8 column using a gradient of water/acetonitrile (both containing 0.1% trifluoroacetic acid or 0.2% acetic acid). The pure fractions were determined by analytical HPLC, on a Vydac Ci8 column using a gradient of water/acetonitrile (both containing 0.1% trifluoracetic acid) and pooled together and lyophilized to give flaky white solids. 8. Peptides were analyzed by analytical reverse phase HPLC on a Vydac Ci8 column using a gradient of water/acetonitrile (both containing 0.1% trifluoroacetic acid), and fast atom bombardment mass spectroscopy. Example 9 B Receptor Antagonist Compound Synthesis The B i receptor antagonists corresponding to the compounds prepared in Example 8, wherein the C-terminal arginine is deleted, are prepared in an analogous manner. The solid phase synthesis is initiated using Boc-Oic-PAM resin obtainable from a commercial supplier (Advanced Chemtech, Louisville, KY). Example 1 Bradykinin Binding Procedures Guinea Pig Ileum Binding Binding of 3H-bradykinin was performed using the method of D.C. Manning, R. Vavrek, J.M. Stewart, and S.H. Snyder, J. Pharmacol. Exp. Ther.. (1986) 222:504. The tissues used in the binding assay were terminal ileum from male Hartley guinea pigs (150-350 g). After dissection, tissues were placed in 20 volume of ice-cold buffer A (25 mM TES containing 0.2 g/1 of 1,10-phenanthroline adjusted of pH 6.8 with ammonium hydroxide) and homogenized using a Ploytron Tissumizer at setting 6 for 15 seconds. The homogenate was centrifuged at 50,000 x g for 10 minutes, the supernatant discarded, and the pellet resuspended in ice-cold buffer A by homogenization with the Polytron. Each tissue was homogenized and centrifuged three times. The final pellet was resuspended in buffer A containing bovine serum albumin (1 g/1) and Bacitracin (0.14 g/1) to a final volume of 170 ml/g of the original tissue weight. The binding assay consisted of 1 mM in 12 x 75 mm polyproproylene tubes: 50 μl 3 H-bradykinin (20,000 dpm, -0.3 nM in the final assay volume), 100 μl displacing drug in buffer A, and 750 μl tissue homogenate. Each tray contained tubes, to which no drug was added to measure maximum binding and tubes to which bradykinin (1 μM final concentration) had been added, to measure specific binding. Specific binding accounted for 96-98% total binding. Tubes were incubated for 90 minutes at ambient temperature. The assays were terminated by filtration over Whatman GF/B glass fiber filters that had been pretreated for 2 hours with polyethyleneimine (2 g/1) using a Brandel Tissue Harvester, followed by washing with 4 x 1 ml aliquots of ice-cold 50 mM Tris, pH 7.4. Filters were dissolved in Ready-Safe Fluor (Beckman) for at least 90 minutes before quantitation by liquid scintillation spectrometry. K_d values were determined using saturation binding and analysis by EBDA (G.A. MacPherson, J. Pharmacol. Methods (1985) 213), followed by LIGAND (PJ. Munson, D. Rodbard, Anal. Biochem. (1980) 220). Ki values were determined using competitive analysis followed by EBDA and LIGAND. Human Bradykinin Receptor Binding The human bradykinin B2 receptor was cloned by Hess et al. ( Biochem. Biophvs. Res. Comm. (1992) 184:260-268). A human bradykinin B2 receptor was expressed in CHO/K cells. Briefly, approximately 2 x 10^ plaques from a human uterus λ gtlO cDNA library (Clontech Laboratories; Palo Alto, CA) were screened using a PCR fragment containing the coding region of the rat n B2 receptor. This probe was generated by random-primed synthesis in the presence of α[32p]dATP. Duplicate filters were hybridized overnight at 42°C in 1M NaCl, 50 mM Tris pH 7.5, 5 X Denhardt's, 200 μg/ml salmon sperm DNA, 1% SDS, and 20% formamide. The filters were washed at 65°C in 1 X SSC and 1% SDS. Coincident positively hybridizing plaques were purified and rescreened j 5 with the same probe and stringency conditions. EcoRI fragments of positive clones were inserted into Bluescript/KS II + vector (Stratagene; La Jolla, CA) for sequence determination. The nucleotide sequence of the cloned human B2 receptors was determined using double-stranded DNA and the dideoxy chain termination 2 Q method. Commercially available T3 and T7 oligonucleotides (USB; Cleveland, OH) and synthetic 21-mer oligonucleotides (DNA/RNA Synthesizer; Applied Biosystems Inc.; Palo Alto, CA) from both the known rat sequence and the determined human sequence were used to identify the nucleotide sequence from the 5' untranslated end to the BgUI site in the 3' untranslated end of the clone. 25 The Hindm Xbal fragment of one full-length clone, 126A, was inserted into pcDNAI neo vector (Invitrogen; San Diego, CA) for expression in mammalian cell lines. CHO/K cells were plated in 2 ml of growth medium (Ham's F12 with 10% FBS) per 6 well plate and incubated at 37°C, 5% CO2 until they were 60% 3 confluent. For each well, 4, 12, and 16 μg of DNA was diluted in 100 μl Opti-MEM I reduced serum medium (Gibco/BRL; Gaithersburg, MD). 12 μl of TransfectASE reagent (Gibco/BRL) was diluted in a separate aliquot of 100 μl Opti-MEM I. The DNA and TransfecASE solutions were combined, mixed gently, and incubated at 25°C for 15 minutes. This solution was then diluted to 35 1 ml with Opti-MEM I. Each well was washed twice with Opti-MEM I and 1 ml of the DNA/TransfectAse complex was added to each well. After a 5 hour incubation at 37°C and 5% CO2, 1 ml of Ham's F12 with 20% FBS was added to each well and cells were incubated overnight. Media was replaced with growth medium and incubated for additional 24 hours. Cells were harvested by trypsinization and replated in selection medium (Ham's F12, 10% FBS and 500 μg/ml Geneticin (Gibco/BRL). Media was replaced every 48 hours for 2 weeks. Any colonies remaining after selection were transferred to separate 10 cm dishes, grown to confluency and positive clones were determined by binding of 3H-NPC17731 a bradykinin antagonist peptide described by Burch et al. (DuPont Biotech update (1992) 4:127-140). Colonies expressing the receptor were put out at limiting dilution. Cells were expanded and positive clones were identified as above. A cell line designated H2O.2 was used to quantitate binding of the compounds of the invention to the human bradykinin B2 receptor. Radioligand Binding Assays This assay measures the ability of a bradykinin antagonist compound to compete for binding to the human B2 receptor with tritiated bradykinin or tritiated NPC17731, a bradykinin analog which is known to bind the B2 receptor and act as a bradykinin antagonist. H2O.2 cells were grown to confluency in Ham's F12 media containing 10% FBS and 500 μg/ml Geneticin. Growth media was aspirated and the monolayer washed once with Dulbecco's PBS without Ca*" 1" and Mg ++ . Cells were scraped in Dulbecco's PBS and centrifuged at 2,000 x g for 10 minutes. Pellets were resuspended in 25 mM TES, 1 mM 1,10-phenanthroline pH 6.8 buffer and homogenized using a Ploytron at setting 5 for 10 seconds. An aliquot was taken for a protein determination using a BioRad protein assay kit. Membranes were centrifuged at 48,000 x g for 10 minutes at 4°C. Pellets were resuspended in the TES buffer with 0.1% BSA and 0.014% bacitracin. 0.5 ml aliquots were frozen in liquid N2 and stored at -80 β C for up to 2 weeks. Membranes from H2O.2 cells previously prepared were thawed at 37 β C and diluted in 25 mM TES, 1 μM 1,10-phenanthroline pH 6.8 containing BSA and bactiracin. For saturation binding assays, increasing concentrations of 3 H-bradykinin ( 3 HBK) or 3 H-NPC 17731 (a known bradykinin antagonist which binds the B2 receptor and which has the amino acid sequence)were incubated with 16.5 μg of membrane protein in a total volume of 3 ml of the same buffer. Non-specific binding was determined with 1 μM bradykinin. The tubes were incubated 90 minutes at 25°C and the assay was terminated by rapid vacuum filtration onto Whatman GF/B filters presoaked with 0.2% PEI for 3 hours followed by 2 X 3 ml aliquots of ice-cold 50 mM Tris, pH 7.4. Radioactivity was counted with a Beckman scintillation counter. Whole Cell Binding Assay Binding to confluent monolayers of transfected cells expressing the human B2 receptor (7,000 fmol/receptor/mg) grown in 24-well plates was measured by incubating cells in 1 ml cell binding buffer (PBS/Ca/Mg, 1% BSA, 0.14 mg/ml bacitracin) containing 10'IOM 3H-bradykinin and various concentrations of unlabeled analogs for 4 to 10 hours at 4 β C. Cells were washed three times with cell binding buffer and lysed in 0.1N NaOH. An aliquot of the lysate was counted to measure radioactivity bound. Example 11 Guinea Pig-Ileum Contraction Assay Guinea pig intestine was removed and placed in a Petri dish containing Tyrodes solution and cut into 3-4 cm segments. The longitudinal muscle was separated from the underlying circular muscle using a cotton applicator (Paton and Zar, J. Phvsiol. (1968) 124:13. Muscle strips were connected to isometric force-displacement transducers (Grass or Gould) coupled to a physiograph and placed in tissue baths containing Tyrode's solution at 37°C. Each preparation was suspended under a resting tension of 2 g. After equiibration of the tissues, appropriate volumes of bradykinin solutions were cumulatively added to the 10 ml tissue baths to increase the concentration of bradykinin in the bath step-by-step without washing out after each single dose. Higher concentrations were added only after the preceding contraction had reached a steady value. When the next concentration step did not cause a further increase in contraction, it was assumed that the maximum effect had been obtained and the tissue was washed to remove bradykinin and allowed to recover for 15 minutes. Antagonism of bradykinin responses to the presence of antagonist were determined by repeating the cumulative addition procedure for bradykinin after the tissue has been exposed to the antagonist for 5 minutes. Three or four different concentrations of antagonist were studied sequentially in the same preparations. Responses were expressed as a percentage of the maximum concentration elicited by bradykinin in the absence of antagonist. pA2 values were calculated by Schild analysis. Assay Remits Table I lists results obtained in the various assays described above for a variety of bradykinin antagonist compounds of the invention. In addition the atomic volume, in cubic angstroms, has been calculated for the Y moieties of a number of the compounds, according to the previously described method of Pearlstein, and is given in Table I. Ul t O COMPOUND o 496±92 H-D-Aig-Arg-NH TABLE 1 (conL) Radioligand Binding Guinea Pig Whole Ileum Atomic Cell Human Human Guinea Contraction Volume COMPOUND 3 HBK 3 HBK 3 H17731 Pig PA, _ Ul H-D-Aig-Arg-Pro-N 15 174 Phe-Ser-D Hype(tran$-thiophenyl)-Oic-Arg-OH TABLE 1 fcont) Radioligand Binding Guinea Pig Whole Ileum Atomic Cell Human Human Guinea Contraction Volume COMPOUND 3 HBK 3 HBK 3 H17731 Pig PA, A 3 7.7 6.7 H-D-Arg-Arg-X[Ph]-CH2C0-Ser-D-Tic-0ic-Arg-0H 6.3 H-D-Arg-Arg-X[4-CH3Ph]-CH2CO-Ser-D-Tic-Oic-Arg-OH 6.8 H-D-Arg-Aιg-X[c-C6HπCH2]-CH2CO-Ser-D-Tic-Oic-Arg-OH 0.73 6.4 H-D-Arg-Arg-Xtc-C6Hιι]-CH2CO-Ser-D-Tic-Oic-Aιg-OH 5.80 H-D-Arg-Arg-X[n-Pr]-CH2C0-Ser-D-Tic-0ic-Arg-0H H-D-Arg-Aιg-X[4-CH3θPh]-CH2CO-Ser-D-Tic-Oic-Arg-OH 18.6 H-D-Arg-Arg-X[Phl-CH2C0-Ala-D-Tic-0ic-Arg-0H 66 H-D-Arg-Arg-X[Ph]-CH2C0-Gly-D-fic-0ic-Arg-0H 54 H-D-Arg-Arg-X[Rι]-CH2C0-N-BnGly-D-Tic-0ic-Arg-0H Oι OI o COMPOUND H-D-Arg-Aιg-X[Ph]-CH2C0-N-BnGly-Ser-D-Tιc-0ic- H-D-Arg-Aτg-X[Ph]-(CH2)4C0-D-Tic-0ic-Arg-0H

Claims: Oi to t Ol O Ol o Ol TABLE 1 (cont.) Radioligand Binding Guinea Pig Ileum Atomic Guinea Contraction Volume COMPOUND Pig PA, A 3 Example 12 Determination of BI receptor antagonist activity The ability of a pseudopeptide of the invention to antagonize B i receptor mediated inflammatory responses in vivo can be confirmed by testing the ability of the pseudopeptide to inhibit des Arg 9 -bradykinin induced hypotension in rabbits that have been pretreated with lipopolysaccharides. It is known that, in the presence of inflammation-inducing substances, including lipopolysaccharide and interleukin-1, Bi receptors are upregulated and respond to des Arg9-bradykinin to produce inflammatory responses, including hypotension. Male New Zealand white rabbits (1.5-2.0 kg) are pretreated with a freshly made solution of LPS (10 μg/100 ml) 5 hr prior to anesthetizing with sodium phenobarbital i.v. The left carotid artery is cannulated for recoding mean arterial blood pressure and the left jugular vein for des Arg^-bradykinin (1 μg/kg) and the pseudopeptide to be tested. Animals are pulsed with a bolus of des Arg9-bradykinin (3x) at 5 minute intervals to produce a basal hypotensive response. Test pseudopeptide is then administered as a bolus prior to des Arg9- bradykinin and its ability to antagonize the Bi -mediated hypotensive response is determined as % inhibition. CLAIMS 1. A compound having the formula X-Y-Z wherein: X is a moiety having a net positive charge selected from the group comprising a positively charged amino acid and an organic group; Y is a hydrophobic organic moiety having the following characteristics: a. a nitrogen atom at the X-Y junction; b. a carbonyl group at the Y-Z junction; c. the hydrophobic organic moiety between the nitrogen atom and the carbonyl group is selected from the group consisting of a carbocyclic, heterocyclic and a linear organic moiety; d. an atomic volume in the range of 135 A to 300 A3; e. an allowed conformation such that an end-to-end distance between the flanking nitrogen and carbonyl atoms is about 5.0 A ± 1.5 A; f. with the proviso that Y cannot consist of naturally occuring amino acids; Z is an arrangement of atoms which inherently adopt a beta turn conformation and has a positive charge near the distal end; said compound having the ability to specifically compete with native bradykinin for binding to the bradykinin B2 receptor; and pharmaceutically acceptable salts thereof. 2. The compound of Claim 1 wherein: X is a mono-, di- or tri-peptide independently selected from the group consisting of the L- and D-isomers of Arg, Gin, Asn, Lys, Sar, N-e-acetyl-Lys, NG-p-tosyl-Arg, NG-nitro-Arg, acetyl- Arg, and citrulline; and Z has the formula E-F-G-H-Cn, wherein E is a direct bond or selected from the group consisting of Ser, Thr, Gly, N-BnGly, Val, Ala, Cys and Tyr; F is selected from the group consising of a D-aromatic amino acid and a D-Hype; G is selected from the group consisting of Oic, Aoc, Thz, octahydro-lH- isoindole-1 -carboxylic acid, pipecolinic acid, Pro, 4Ηyp, azetidine- 2-carboxylic acid, Aib, Leu, He, Val, an aromatic amino acid that is Phe, Thi, Tic, indoline-2-carboxylic acid, homoPhe, Trp, Tyr, Nal, and a Hype; H is selected from the group consisting of Arg, Orn, Asn, Gin, and Lys; Cn is a hydroxyl group or a C-terminal extension selected from the group consisting of carboxamido, alkoxy, an acidic, basic or neutral aliphatic, aromatic, or cylic amino acid residue of the D- or L- configuration. 3. A compound of Claim 2 wherein: X is D-Arg-Arg; E is a direct bond or is selected from the group consisting of Ser, Gly and Val; F is selected from the group consisting of D-Phe, D-Tic and a D-Hype; G is selected from the group consisting of Phe, Oic, Aoc, Tic and a Hype; H is Arg; and Cn is selected from the group consisting of a hydroxyl group, an amide group and an alkoxy group. A compound of Claim 1 wherein Y has the formula: A compound of Claim 1 having the formula: -Tic-Oic-Arg-OH 6. A compound of Claim 1 wherein Y has the formula: 7. A compound of Claim 1 having the structure: 8. A compound of Claim 1 wherein Y has the formula: A compound of Claim 1 having the formula: 10. A compound of Claim 1 wherein Y has the formula: 11. A compound of Claim 1 having the stucture: 12. A compound of Claim 1 wherein Y has the formula: O 13. A compound of Claim 1 wherein Y has the formula: 14. A compound of Claim 1 wherein Y has the formula: 15. A compound of Claim 1 wherein Y has the formula: 16. A compound of Claim 1 wherein Y is a C6 to C18 olefinic aminoalkenoyl having the following formula -NH-(CH 2 ) m -R 5 -(CH 2 ) n -R 6 (CH 2 )o-C- wherein: R5 and R > are independently selected from the group consisting of a direct bond, a C3 to C8 carbocycle, and a C2 to Ci8 monoolefin or a C4 to Ci8 polyolefin containing 2 to 5 double bonds which may optionally be incorporated into a cyclic system, m, n, and 0 are independently 0 through 12, with the proviso that their total does not exceed 16. 17. A compound of Claim 16 wherein: X is D-Arg-Arg and Z is Ser-D-Tic-Oic-Arg 18. A compound of Claim 1 wherein Y has the formula: R 7 0 -R 3 -R 4 -(CH 2 ) m -R 5 -(CH 2 )„-R 6 -(CH 2 ) o -CH-C- wherein: R 3 is selected from the group consisting og 2-pyrrolidinyl, Pro, 4Hyp, Oic, dehydroPro, Tic, Aoc, L-azetidine-2-carboxylic acid, Eac, Gly, Thz and Aib; R 4 is selected from the group consisting of a direct bond and an imino group; R 5 and R 6 are independently selected from the group consisting of a direct bond, a C3 to Cδ carbocycle and a C2 to Ci8 monoolefin or a C4 to Cis polyolefin containing 2 to 5 double bonds which may optionally be incorporated into a cyclic system; R 7 is selected from the group consisting of hydrogen, hydroxymethyl, Ci to s alkyl, benzyl, thiophenylmethyl and furanylmethyl; and m, n, and o are independently an integer from 0 through 12, with the proviso that their total does not exceed 12. 19. A compound of Claim 18 wherein: X is D-Arg-Arg and Z is Ser-D-Tic-Oic-Arg. 20. A compound of Claim 18 wherein: X is D-Arg-Arg-Pro and Z is Ser-D-Tic-Oic-Arg. 21. A compound of Claim 18 wherein: R 5 is selected from the group consisting of a direct bond, a C3 to C% carbocycle, and a C2 to Cis monoolefin or a C4 to Cis polyolefin containing 2 to 5 double bonds which may optionally be incorporated into a cyclic system; R 6 is selected from the group consisting of a direct bond and a C2 to Cis monoolefin; R 7 is selected from the group consisting of hydrogen and benzyl; and m, n, and 0 are independently an integer from 0 to 6. 22. A compound of Claim 1 wherein Y has the following formula: wherein R5 and R6 are independently selected from the group consisting of a direct bond, a C3 to C8 carbocycle, and a C2 to Ci8 monoolefin or a C4 to Ci8 polyolefin containing 1 to 5 double bonds which may optionally be incorporated into a cyclic system; R 7 is selected from the group consisting of hydrogen, hydroxymethyl, Cl to C6 alkyl, benzyl, thiophenylmethyl and furanylmethyl; m, n, and o are independently 0 through 12, with the proviso that their total does not exceed 12. 23. A compound of Claim 22 wherein: X is D-Arg-Arg or D- Arg-Arg-Pro and Z is -Ser-D-Tic-Oic-Arg. 24. A pharmaceutical composition useful as a bradykinin receptor antagonist comprising a pharmaceutical carrier and an effective amount of the peptide of Claim 1. 25. A pharmaceutical preparation for treating local pain and inflammation from bums, wounds, cuts, rashes or other trauma and pathological conditions caused by the production of bradykinin or related kinins by an animal, which comprises an effective amount of the peptide of Claim 1 to antagonize bradykinin and a suitable pharmaceutical carrier. 26. A process for treating local pain and inflammation, which comprises administering an effective amount of the peptide of Claim 1 to an animal in need thereof. 27. A process for antagonizing bradykinin receptor activity in mammals, which comprises: administering to a mammal an effective amount of the compound of Claim 1 to antagonize bradykinin receptor activity. 28. A compound having the formula: X-Y-Z' wherein, X is a moiety having a net positive charge selected from the group comprising a positively charged amino acid and an organic group; Y is a hydrophobic organic moiety having the following characteristics: a. a nitrogen atom at the X-Y junction; b. a carbonyl group at the Y-Z' junction; c. the hydrophobic organic moiety between the nitrogen atom and the carbonyl group is selected from the group consisting of a carbocyclic, heterocyclic and a linear organic moiety; d. an atomic volume in the range of 135 A3 to 300 A3; e. an allowed conformation such that an end-to-end distance between the flanking nitrogen and carbonyl atoms is about 5.0 A ± 1.5 A; f. with the proviso that Y cannot consist of naturally occuring amino acids; Z' is an arrangement of atoms which inherently adopt a beta turn conformation and lacks a positive charge near the distal end; and pharmaceutically acceptable salts thereof. 29. A method of treating a condition characterized by chronic inflammatory hyperalgesia in a mammal requiring such treatment which comprises administering an effective amount of a compound of Claim 28 to such mammal.

Inventors: KYLE DONALD JAMES, LU ZHIJIAN, MAVUNKEL BABU JOSEPH, KYLE, DONALD, JAMES, LU, ZHIJIAN, MAVUNKEL, BABU, JOSEPH
Applicants: KYLE DONALD JAMES, LU ZHIJIAN, MAVUNKEL BABU JOSEPH, SCIOS NOVA INC, KYLE, DONALD, JAMES, LU, ZHIJIAN, MAVUNKEL, BABU, JOSEPH, SCIOS NOVA INC.
Application year: 1994
Main classification: C07K7
IPC codes: A61K31, A61K38, A61P25, A61P29, A61P43, C07D215, C07D221, C07D471, C07K7, G01N33